Compositions and methods for treatment of candidiasis

ABSTRACT

The present invention relates to novel oral compositions and vaccines, and in particular to oral vaccines for the prevention or therapy of candidiasis.

TECHNICAL FIELD

[0001] The present invention relates to novel oral vaccines and inparticular to oral vaccines for the prevention or therapy ofcandidiasis.

BACKGROUND

[0002]Candida albicans is a yeast-like dimorphic fungus that coloniseshuman mucosal surfaces of the mouth, vagina and the gastrointestinaltract as part of the normal microbial flora. It is also an opportunisticpathogen which may trigger a local mucositis such as stomatitis andvaginitis, or it may invade to become a systemic infection.

[0003] It is clear that the various outcomes reflect different balancesof a host-parasite relationship. While mechanisms of protection are notentirely understood, cellular mechanisms (in particular the Tlymphocyte-macrophage unit) are thought to be important in containingboth local and systemic spread of the microbe.

[0004] The clinical spectrum of mucositis includes:

[0005] Recurrent/Persistent Stomatitis.

[0006] This is a common problem in the elderly, particularly those withdental prosthesis (dentures). In the latter, about two thirds developmucositis in relationship to colonisation with C. albicans within theplaque that accumulates on the prosthesis. It is of interest that thissituation represents low antigen load and possibly involves a particularhost response. Similar fungal problems can be seen in younger subjects,usually wearing prostheses. In subjects with cellular immune deficiency(especially those with HIV disease) persistent C. albicans-relatedstomatitis (or oral thrush) is a common and significant complication.Subjects using inhaled steroids (usually for asthma) commonly developoral thrush due to a downregulation of mucosal defence by the steroid.

[0007] Recurrent Vulvovaginal Candidiasis.

[0008] This is a common problem in women (3-5%) occurring at the time ofthe menstrual cycle of oestrogen withdrawal (pre-menstrual) when the“mucosal gate” closes, excluding transport of specific T cells into themucosal tissues. In this group the “T cell pool” is reduced, thus thetenuous hold on restricting C. albicans within the reproductive tract ischallenged during this time, with clinical mucositis the result.

[0009] Oesophagitis.

[0010] This is a common and concerning complication of subjects withimpaired immunity, most usually linked to systemic disease such ascancer or in those taking immune suppressive therapy. It is oftenasymptomatic, following oral thrush, becoming the focus from whichlife-threatening systemic spread originates. Thus an oral vaccinecapable of maximising efficiency of mucosal T cell function, and thusreducing risk of local/systemic disease would be of considerable valuefor prevention (in ‘at risk’ populations) or therapy (of clinicalmucositis) of C. albicans infection of the upper gastrointestinal tract,particularly in immune suppressed subjects would be a primary indicationfor this vaccine.

[0011] Bowel Colonisation.

[0012] It is thought that colonisation of the gut with C. albicanstriggers an extraordinary range of illnesses and symptoms, from chronicfatigue to “total body allergy”. An industry has grown up around“controlling the candida”. It would be advantageous to have a vaccinewhich could reduce the candida load within the gut by several logs, andthus be an attractive therapeutic proposition.

[0013] Other Mucosal Sites and Situations.

[0014] The bronchus can become colonised with C. albicans when damaged,especially following repeated antibiotic administration or with mucosaldamage. Occasional subjects develop colonisation of the lower urinarytract. A more common problem is recurrent or persistent “thrush” at oneor other site following repeated antibiotic use. Such circumstancesprovide a clinical opportunity for a vaccine, especially in apreventative fashion, ie where certain antibiotics are used long term,the risk of C. albicans overgrowh would be reduced by giving a vaccine.

[0015] Therefore, improved vaccines and methods for the prophylacticand/or therapeutic treatment of candidiasis are needed.

[0016] An object of the present invention is to overcome or ameliorateat least some of the disadvantages of the prior art treatments, or toprovide a useful alternative.

SUMMARY OF THE INVENTION

[0017] The present invention is based in part on the unexpected findingthat oral immunisation with the blastococcoid form of Candida albicansprevents infection by the organism or treats established infection.Based on the prior art teaching it would have been expected that theinvasive, mycelial, form would be an optimal immunogen. The presentinvention is also based on the observation that the blastococcoid formof Candida drives the T cell-macrophage unit to secrete particularcytokines, such as EN-γ, and also nitric oxide (NO), into the salivaThis not only creates an environment toxic to the fungus but alsoprevents a switch to the mycelial (invasive) form of the fungus. Theeffect is expected to be similar with respect to all mucosal surfacesand secretions.

[0018] According to a first aspect there is provided a compositionsuitable for oral administration, the composition including inactivatedCandida albicans, for prophylactic or therapeutic treatment of acondition caused by Candida albicans colonisation and/or infection at amucosal surface.

[0019] According to a second aspect there is provided a compositionsuitable for oral administration, the composition includingblastococcoid form of Candida albicans, for prophylactic or therapeutictreatment of a condition caused by Candida albicans colonisation and/orinfection

[0020] Preferably the condition is selected from an oral, nasophayngealor respiratory tract colonisation and/or infection by Candida albicans,

[0021] The composition may be a vaccine which includes either the wholeorganism, inactivated or live but attenuated, a sonicate of the organismor any fragment thereof which includes one or more individual antigens.

[0022] Where the whole organism is considered, the blastococcoid form ofthe organism is particularly preferred but the mycelial form may also beused.

[0023] The compositions of the present invention may also includeconventional pharmaceutical carriers and adjuvants. When an adjuvant isincluded it is preferred that it selected for its ability to induce aTh1 response.

[0024] The compositions of the present invention may also include aprobiotic, preferably a probiotic bacterium. Advantageously theprobiotic bacterium may be selected from lactic acid bacteria and thepreferred bacteria are Lactobaciflus acidophilus.

[0025] According to a third aspect there is provided a method ofprophylactic or therapeutic treatment of a condition caused by Candidaalbicans colonisation and/or infection at a mucosal surface, includingthe administration to a subject requiring such treatment of acomposition according to any one of the previous aspects.

[0026] According to a fourth aspect there is provided a method ofprophylactic or therapeutic treatment of a condition caused by Candidaalbicans colonisafion and/or infection, including the administration toa subject requiring such treatment of a composition according to thefirst or the second aspects.

[0027] Preferrably the condition is mucositis.

[0028] Even more preferred condition is selected from the groupconsisting of recurrent/persistent stomatitis, recurrent vulvovaginalcandidiasis, oesophagitis and lower urinary tract or bowel colonisation.

[0029] The method of treatment according to the present invention mayfurther include administration of one or more adjuvants.

[0030] The adjuvant is preferably selected to induce a classical Th1response (eg BCG) or, where required, a Th2 response(eg B sub-units ofcholera toxin etc) or shortcut vaccines (eg pertussis).

[0031] Adjuvants may either be replaced or supplemented withadministration of probiotics such as , for example lactobacilli. Evenmore preferred are microorganisms of the Lactobaccillus species,particularly Lactobacillus acidophilus. However, other bacteria such asLactobacillus fermentum and Mycobacterium vaccae may also be used.Further, it will be understood that other adjuvants, microorganisms orcomponents thereof, which can drive a Th1 response, would also besuitable. The term “probiotics” as used in the context of the presentinvention is in tended to include in its scope agents which do notnecessarily act as conventional probiotics but will be capable ofinducing a T cell response, or alter the cytokine pattern, as describedherein.

[0032] Probiotics and/or adjuvants may be administered orally orparenterally, and may be given before, during or after cessation oftreatment with the compounds of the present invention. Administration ofprobiotics before, during or after treatment with the compounds of thepresent invention is particularly preferred because of their ability to“skew” the cytokine pattern and thus achieve optimal cytokine balancefor effective treatment.

[0033] According to a fifth aspect there is provided a method formonitoring vaccine requirement or vaccine efficacy including themeasurement of IFN-γ, NO, and/or IL-4.

[0034] According to a sixth aspect there is provided a method ofidentifying Candida isolate and/or Candida antigen effective as vaccineor vaccine component, including the measurement of IFN-γ, NO, IL-12and/or IL-4 in a mouse model.

[0035] For convenience the measurements can be performed on salivasamples however blood samples can also be used as well as tissue samplessuch as lymph nodes and the like. When using lymph nodes or similarlymphoid tissue, assessment can be made on proportion of cells whichexpress the relevant cytokines.

[0036] According to a seventh aspect there is provided a Candida isolateand/or Candida antigen identified by the method of the sixth aspect.

[0037] For convenience the measurements can be performed on salivasamples however blood samples can also be used as well as tissue samplessuch as lymph nodes and the like. When using lymph nodes or similarlymphoid tissue, assessment can be made on proportion of cells whichexpress the relevant cytokines.

BRIEF DESCRIPTION OF FIGURES

[0038]FIG. 1. Patterns of colonisation with C. albicans in BALB/c andDBA/2 mice A (left panel); Mice were infected by swabbing the oralmucosa with C. albicans (1×10⁸ CFU/mL). At various times indicated, thelevel of colonisation was assessed by swabbing the oral cavity. Datashown are mean±SE for 3-5 mice. * p<0.05, *** p<0.001 denotessignificance difference between values from BALB/c and DBA/2 mice. B (right panel): The numbers of blastospore and hyphae in oral tissues werecounted by light microscopy (magnification ×40) after staining with H&Eand PAS stains. Data shown represent mean±SE for 3-5 mice.

[0039]FIG. 2. Lymphocyte proliferation p Cervical lymph node (CLN) cellswere stimulated or unstimulated with Candida antigen for 4 days afterwhich time thymidine incorporation was assessed. The results shown aremean cpm±SE for 3 mice. * p<0.05, ** p<0.01 compared with values fromunstimulated cells.

[0040]FIG. 3. C. albicans-specific serum IgG and IgA antibody

[0041]C. albicans-specific IgG and IgA antibody levels were measured inserum and saliva from infected mice by ELISA. The time 0 representsuninfected mice. The results shown are mean±SE for three mice. *,p<0.05, ** p<0.01 compared with values from BALB/c or DBA/2 mice.

[0042]FIG. 4. IL-4 and IFN-γ mRNA gene expression in CLN cells

[0043] Total RNA were extracted from CLN cells of mice infected with C.albicans and analyzed by RT-PCR using cytokine-specific primers.Equivalent loading of each sample was determined by G3DPH message.

[0044]FIG. 5. IL-4, IL-12 and IFN-γ production by CLN cells stimulatedin vitro

[0045] CLN cells from infected mice were stimulated with C. albicansantigen for 3 days after which time the culture supernatants wereassayed for cytokines by ELISA. The time 0 represents uninfected mice.The results shown are mean±SE for three to five mice. * p<0.05, **p<0.01 compared with values from BALB/c or DBA/2 mice.

[0046]FIG. 6. Effect of treatment with anti-IL-4 antibody on theresistance to acute infection with C. albicans

[0047] BALB/c mice were injected i.p with 30 μg of rat anti-IL-4 or withpurified rat IgG1 matched isotype on days 1, 3 and 5 after challengewith yeasts cells. On various days, the number of yeasts in the oralcavity was determined and the results were expressed as mean colonyforming units (CFU)±SEM for 3-5 mice. *, p<0.05.

[0048]FIG. 7. Clearance of C. albicans in mice immunised orally witheither blastospores (B) or hyphae (H).

[0049]FIG. 8. Effect of oral immunisation on saliva levels of IFN-γ

[0050]FIG. 9. Effect of treatment with L-MLNA on clearance of C.albicans in mice

[0051]FIG. 10. Comparison of oral and subcutaneous administration ofCandida vaccine (Candivax) on immunisation efficacy

[0052]FIG. 11. Oral immunisation with Candida soluble antigens

[0053]FIG. 12. Effect of Candivax on T cell proliferation in response tostimulation with Candida antigens

[0054]FIG. 13. Oral colonisation with Candida albicans after challenge,before and after immunisation

[0055]FIG. 14. Gut colonisation with Candida albicans following oralchallenge, before and after immunisation

[0056]FIG. 15. Effect of co-administration of Lactobacillus and Candivaxon protection against oral candidiasis

[0057] The invention will now be more particularly described withreference to non-limiting examples.

EXAMPLES Example 1 Materials and General Methodology

[0058] Mice

[0059] Unless otherwise stated male BALB/c (H-2d) and DBA/2 mice (H-2d),6-8 weeks old were purchased from the Animal Resource Centre, Perth,Western Australia. They were housed in groups of 3-5 and provided withfood and water adlibitum. All mice were used after one week ofacclimatization.

[0060] Fungal Culture

[0061]Candida albicans isolate 3630 was obtained from the NationalReference Laboratory, Royal North Hospital, Sydney, Australia. The yeastcells were cultured in sabouraud dextrose broth (Oxoid, Hampshire, UK)for 48 hrs at 25° C. in a shaking water bath. The blastospores weretransferred into fresh medium and cultured at 25° C. for a farther 18hrs. The blastospores were collected by centrifugation, washed twicewith phosphate-buffered saline (PBS) and then adjusted to 10⁸blastospores per mL in PBS until use.

[0062] Candida Antigen

[0063] Freshly cultured C. albicans isolate 3630 was resuspended in PBSat 1× 10¹⁰ /ml and then sonicated in an MSE Soniprep set at 10 amplitudefor 30 cycles with intermittent cooling and sonication. The sonicate wascentrifuged for 10 min at 2000 g after which the supernatant wascollected and dialysed against PBS. After protein estimation, thesolution was filtered-steriled and stored in aliquots at −20° C. untiluse.

[0064] Oral Infection

[0065] Mice were anaesthetised by intraperitoneal injection with 75 mlof Ketamine: Xylazil (100 mg/ml: 20 mg/mL). Briefly, 10⁸/ml ofblastospores in PBS were centrifuged at 14,000 g for 5 mins. The pelletwas recovered on a fine-tip sterile swab (Corsham, Wiltshire, UK) whichwas then used for oral inoculation by topical application into the oralcavity.

[0066] Quantitation of Oral Infection

[0067] Unless otherwise stated, groups of mice (3-5 per group) weresacrificed at various time points to determine the number of C. albicansin the oral mucosa. The oral cavity (i.e. cheek, tongue and softpalate), was completely swabbed using a fine-tipped cotton swab. Afterswabbing, the cotton end was cut off and then placed in an eppendorftube containing 1 ml of PBS. The yeast cells were resuspended by mixingon a vortex mixer before culture in serial 10-fold dilutions onSabouraud dextrose agar (Oxoid, UK) supplemented with chloramphenicol(0.05 g/L) for 48 hrs at 37° C. For histological studies, oral tissueswhich were fixed in 10% formalin and embedded in paraffin. Tissuesections 5 mm thick were cut, mounted on glass slides and then stainedwith haematoxylin and eosin (H&E) or PAS stain for fungi. The numbers ofblastospores and hypahe forms were enumerated by light microscopy. Theresults were expressed as the mean count of five fields at 40 ×magnification.

[0068] Cell Separation and Flow Cytometry

[0069] The cervical lymph nodes (CLN) were excised from 3-5 C.albicans—infected mice for each time point after infection, and singlecell suspensions were prepared (17). Pooled CLN populations wereanalysed in two-colour mode using Lysis 2 software and FASCan cytometry(Bectin-Dickinson, Mountain View, Calif.). The MAbs used for stainingwere fluorescein isothiocynate (FITC)-conjugated (H129.19 anti-CD4 andH57-597 anti-a/b TCR) or phycoerythrin (PE)-conjugated (H53-6.7anti-CD8a, ID3 anti-CD19 and GL3 anti-a/d TCR). FITC- or PE-conjugatedisotype-matched antibodies were used as negative controls. All Mabs werepurchased from Pharmingen. At least 10,000 viable cells were used fromeach preparation for analysis.

[0070] Lymphoproliferation Assay

[0071] Pooled CLN cells in RPMI 1640 medium supplemented with 10% FCSwere cultured in triplicate at 0.2×10⁶ cells per well in wells of a96-well round bottomed microtitre plate (Nunc, Denmark). C. albicansantigen was added to each well at a final concentration of 2.5 mg/mL.The cultures were incubated for 72 hrs in an atmosphere of 5% CO₂ in ahumidified incubator. Thymidine incorporation was measured by pulsingthe cells with 1 μCi of ³H-labelled thymidine (Amersham, Aylesbury, UK)for the final 6 hrs of incubation before harvesting and counting. Theresults were expressed as mean cpm±SEM.

[0072] Antibody Assay

[0073] A microplate ELISA assay was used to quantitate specific antibodyin the saliva and serum. Immunopolysorb microtiter (Nunc, Denmark) wellswere coated with 50 μg/mL of C. albicans antigen in 0.1 M sodiumborate-buffered saline (pH 8.4). Appropriate serial dilutions of theserum and saliva samples were added to each well. Bound antibodies weredetected by the addition of biotinylated goat anti-mouse IgG or IgA(Sigma-Aldrich) and followed by alkaline phosphatase-conjugatedstreptavidin (AMAAD, Australia). After addition of the substratesolution, the optical density of duplicate samples was read at 450 nmwith an ELISA plate reader (BioRad, Richmond, Va.).

[0074] RT-PCR

[0075] RNA extraction and amplification of synthesised cDNA fromlymphoid cells have been described (31,42). RNA extraction andamplification of synthesised cDNA from lymphoid cells have beendescribed (31,42). Briefly, 10 mL of total RNA extracted from 4×10⁶/mLof CLN cells was added to 20 mL of RT mix containing 6 mL of 5× RTreaction buffer (250 mM Tris-HCl, 375 mM KCl and 15 mM MgCl,), 3 mL of100 mM dithiothreitol, 1.5 mL of deoxynucleotide (10 mM), 1 mL of RNAseinhibitor (40 U/mL), 0.5 mL of MMLV-RT (200 U/mL), 3 mL of oligo-(dT)15,3 mL of acetylated BSA (1 mg/mL) and 2 mL of DEPC-treated water. ThecDNA synthesis was carried out at 42° C. for 1 hr followed by heating at72C for 10 mins. PCR amplification was carried out by adding 5 mL of thefirst strand cDNA to the PCR mix containing: 1 mM of each primer (20mM), 1 mL of 4 mM dNTP mix, 5 mL of 10× PCR buffer, 1.2 mL of 1.5 mMMgCl₂, 0.2 mL Taq DNA polymerize (50 U/mL), and 31 mL of DEPC treatedwater. The mixture was subjected to amplification using a thermal cyclerHybaid, Middlesex, UK) set at 94° C. for 1 min (IL-4 and G3DPH) and 30secs for IFN-g; 600 for 2 mins (IL-4 and G3DPH) and 62C for 1 min(IFN-g), and 72° C. for 3 mins (IL-4 and G3DPH) and 90 secs for IFN-γwith final elongation step at 72° C. for 10 mins. PCR amplification wascarried for 35-40 cycles. PCR fragments were separated on a 2% agarosegel electrophoresis, stained with ethidium bromide and then viewed undera UV transilluminator. The primer sequences were as follows: IL-4, senseGAA TGT ACC AGG AGC CAT ATC; antisense CTC AGT ACT ACG AGT ATT CCA;IFN-g, sense TCT CTC CTG CCT GAA GGA C; antisense ACA CAG TGA TCC TGTGGA A. The amplified DNA products for IL 4 and IFN-γ were 399 bp and 460bp, respectively.

[0076] Cytokine Assay

[0077] CLN cells in RPMI 1640 medium supplemented with 10% FCS werecultured at 4×10⁶ cells per well in the presence of 2.5 mg/mL of C.albicans antigen in a 24 well plate for 3 days (as described above). Theculture supernatants were collected and then assayed for IL-4, IL-12 andIFN-γ by ELISA using matched-antibody pairs and recombinant cytokines asstandards (Pharmingen, San Diego, Calif.). Briefly, immuno-polysorbmicrotitre plates (Nunc, Denmark) were coated with capture ratmonoclonal anti-IL-4 (IgG1), IL-12 (IgG2a) or IFN-γ (IgG1) antibody at 1μg/mL in sodium bicarbonate buffer (pH 8.4) overnight at 4° C. The wellswere washed and then blocked with 1% BSA before the culture supernatantsand the appropriate standard were added to each well. Biotinylated ratmonoclonal anti IL-4, IL-12 or IFN-γ antibody at 2 mg/mL was added asthe second antibody. Detection was done with streptavidin peroxidase(AMRA, Melbourne, Australia) and TMB (Sigma-Aldrich). The sensitivity ofthe cytokine ELISAs was 31 pg/mL. The results were expressed as netCandida-induced counts from which the background was subtracted.

[0078] Infection and Treatment with Anti- IL-4 Monoclonal Antibody

[0079] Mice were injected i.p. with 30 μg rat anti- rIL-4 (31) (clone11B11, Pharmingen, San Diego, Calif.), or with the purified rat IgG1matched isotope in 200 mL of PBS per mouse at days 1, 3 and 5 after oralinfection with 10⁸ yeast cells (C. albicans). The number of yeast in theoral cavity was determined as described above.

[0080] Statistical Analysis

[0081] The data were compared using the non-parametric Mann-WhitneyU-test. P values <0.05 were considered significant. All calculationswere performed using a statistical software program (StatView; AbacusConcepts, CA).

Example 2 Kinetics of Oral Infection by Candida albicans in BALB/c andDBA/2 Mice

[0082] The oral mucosa of BALB/c mice was infected with 10⁸ Candidaalbicans blastospores (blastoccoid form)on day 0,after which time thelevel of colonisation was examined over 28 days. As shown in FIG. 1 (left panel), the levels of colonisation 6 hrs after infection weresimilar in both BALB/c and DBA/2 mice. However, resistance to infectionin BALB/c mice was evident at day 2 after an initial reduction incolonisation at day 1 after inoculation, when compared with a 1-logincrease in the number of yeast in DBA/2 mice (p<0.05). While there wasa decrease in colonisation in BALB/c and DBA/2 mice on day 4, a 2-logincrease in the number of yeast occurred on day 6 in DBA/2 mice(p<0.001), compared with BALB/c mice. By day 8, the BALB/c mice had noyeast in the oral cavity whereas in DBA/2 mice the number of yeast wasabove 3 logs which gradually declined to background level by day 15.Cultures of fecal pellets from mice after inoculation of Candidaalbicans showed no growth or <3 CFU per fecal pellet, thus excluding thepossibility that the repeat cycle of infection in DBA/2 mice was due tocaprophagia.

[0083] To determine whether the pattern of infection was characterisedby different morphological forms of Candida albicans, the proportions ofblastospores and hyphae forms in oral tissues were enumerated. FIG. 1 (right panel) represents the ratios of blastospores to hyphae forms ofCandida in tissue sections of the oral mucosa in BALB/c and DBA/2 mice.After inoculation, the ratios of blastospores to hyphae forms were aboutsimilar in DBA/2 and BALB/c mice. By day 2, there were more hyphae formsthan blastospores in DBA/2 than in BALB/c mice. On day 4, about equalratios of blastospores and hyphae forms were detected in both micestrains. In BALB/c mice, the ratios of blastospores to hyphae continuedto rise over time when 100% of yeast present in the oral mucosa on day 6were blastospores before they were cleared by day S. In marked contrast,a low blastospore to hyphae ratio was detected in DBA/2 mice on day 6and then rising to day 10 before the yeast, consisting predominantly ofblastospores, were cleared on day 15.

Example 3 Cellular Response in the CLN

[0084] The mean number of cells recovered from the CLN increased from9.8×10⁶ to 22×10⁶ cells, and 9.5×10⁶ to 18×10⁶ cells, per mouse 4 daysafter infection with C. albicans in BALB/c and DBA/2 mice, respectively(Table 1). A drop in cell counts on day 6 followed the clearance of C.albicans in both BALB/c and DBA/2 mice, but in DBA/2 mice it wasfollowed by a rise in cell counts after re-infection before decline onday 15. While the relative proportions of CD19+ B cells and the variousT cell subsets remained constant, there was a significant increase inthe percentage of γ/δ T cells above the background level during thecourse of infection. In BALB/c mice, the number of γ/δ T cells increasedby 5-6 fold on day 6 and then declined thereafter when the infection wascleared. In contrast, in DBA/2 mice, increase in the numbers of γ/δ Tcells was cyclical with maximum levels occurring on days 4 and 8 beforefalling to background levels on day 28, when the infection was cleared.

[0085] In Vitro Stimulation of CLN Cells

[0086] The effect of C. albicans colonisation on T cell proliferationwas determined in culture of CLN cells stimulated with C. albicansantigens. As shown in FIG. 2, there was a significantly higherantigen-stimulated T cell proliferative response which peaked at day 4(p<0.05) and day 10 (p<0.05) in DBA/2 mice compared to unstimulatedcontrols. In contrast, a lower ( but significant) increase in theproliferative response in BALB/c mice occurred at day 4 (p<0.05) and wasmaintained thereafter at a similar level after a peak response at day 6(p<0.01). The proliferative response in DBA/2 mice however continued todecline to control levels by day 28.

Example 4 Serum and Local IgG and IgA Antibody Responses

[0087] As shown in FIG. 3, an increase in serum IgG antibody levels wasdetected in both BALB/c and DBA/2 mice 10 days after infection withmaximum levels detected on day 15. The levels of IgG antibody weresignificantly higher in BALB/c mice compared to DBA/2 mice at days 10and 15 (p<0.05) and at day 28 (p<0.01). Similarly, significantly higherlevels of IgA antibody were detected in saliva of BALB/c mice comparedto DBA/2 mice at all time points from day 8, with maximum levels at day15 (p<0.05), before dropping at day 28 (p<0.05).

EXAMPLE 5 Effect of Infection on IL4 and IFN-y mRNA Gene Expression

[0088] The effect of colonisation on mRNA expression of IL-4 and IFN-γin CLN cells was examined by RT-PCR. As shown FIG. 4, IL-4 geneexpression was detected on day 2 in BALB/c mice whereas it was notexpressed until day 6 in DBA/2 mice. While IL-4 gene expressiondisappeared by day 10 in BALB/c mice, it continued to be expressed inDBA/2 mice at day 15. In contrast, IFN-γ mRNA gene expression was firstdetected at 6 hrs after infection and then gradually declined in BALB/cmice, whereas it continued to be strongly expressed in DBA/2 mice overthe 28 days.

[0089] IL-4 , IL-12 and IFN-γ production by CLN cells stimulated with C.albicans antigen. To determine the pattern and the kinetics of cytokineproduction following infection, CLN cells were stimulated with C.albicans antigen for 72 hrs after which time levels of IL-4 and IFN-γ inthe culture supernatants were measured. TABLE 1 γ/δ T cells post primaryinfection in BALB/c and DBA/2 mice (Day 0 represents uninfected mice.The various phenotypes were expressed as percentages of total leukocytesdetermined by FACS analysis. Results shown are mean ± SE for 3-5 mice.)(BALB/c) (DBA/2) LN count LN count Time % γδ (×10⁶) % γδ (×10⁶) 0 0.989.75 0.89 10.47 0.25 0.98 12.03 0.90 10.63 1 1.25 15.47 0.90 13.57 21.65 18.30 2.00 14.83 4 4.25* 21.87 3.54* 16.97 6 6.50** 17.87 2.2012.17 8 3.25* 15.00 4.23* 13.47 10 0.94 17.27 5.68** 14.50 15 0.97 14.471.80 12.33 28 0.88 11.93 0.92 11.93

[0090] Cell numbers expressed as counts per mouse, and the percentagesof γ/δ T cells was significantly different above background. *p<0.05,**p<0.01 compared with background levels.

[0091] As shown in FIG. 5, significantly higher levels of IL-4 wereproduced at day 2 with maximum levels occurring at days 4 and 6 inBALB/c mice than in DBA/2 mice respectively (p<0.01 and p<0.05). Incontrast, an increase in IFN-γ levels was observed in both BALB/c andDBA/2 mice, but with significantly higher levels produced in DBA/2 miceat 6 hrs and at day 2 after infection than was seen in BALB/c micerespectively (p<0.05 and p<0.01). By days 4 and 6, IFN-γ production wasat its highest in BALB/c mice compared to DBA/2 mice where the level ofIFN-γ production was at background levels by day 6 (p<0.01). While theproduction of IFN-γ declined, with exception of a small increase at day15 in BALB/c mice, a marked increase in production was detected in DBA/2mice at days 8 (p<0.05) and 10 (p<0.01). By day 28, the levels of IFN-γreturned to background levels in both mouse strains.

[0092] To determine whether the different levels of IL-4 and IFN-γproduction are related to IL-12 production, CLN cells were isolated atvarious times from BALB/c and DBA/2 mice infected, and then stimulatedwith C. albicans antigen for 3 days, after which IL-12 was measured inthe culture supernatant. As shown in FIG. 5, significantly higherproduction of IL-12 was detected as early as 2 days after infection inDBA/2 mice (p<0.05). In BALB/c mice, an increase in IL-12 production wasdetected at day 6 and day 8. (p<0.05). Following a further increase inDBA/2 mice, IL-12 was then maintained at similar levels for 28 days inboth mice strains.

EXAMPLE 6 Effect of Multiple Injections of Anti-IL-4 Monoclonal Ab onSusceptibility to Candida Infection in BALB/c Mice

[0093] To determine whether the higher production of IL-4 in BALB/c micewas associated with rapid clearance of the yeast, the effect ofanti-IL-4 administration was assessed. FIG. 6 demonstrates that BALB/cmice infected with the yeast followed by administration of 30 μg ofanti-IL-4 on days 1, 3 and 5 after oral infection had a higher carriagerate with a delayed clearance of the yeast compared with untreatedcontrols. However, there was no detectable difference in the amounts ofIFN-γ in CLN cell culture supernatants between anti-IL-4 mAb treated andcontrol C. albicans -infected mice.

EXAMPLE 7 Effect of Immunisaiton by Blastococcoid and Mycelial Forms ofCandida albicans on Clearance of Candida

[0094] DBA/2 mice (n=3-5) were immunised by intragastric intubation with1×10⁹ heat killed C. albicans blastospores or hyphae forms in 0.2 mL PBSevery two days for 18 days. One day after the last immunisation, theoral mucosa of mice was infected with 10⁸ yeast cells by topicalapplication. To compare with systemic immunisation, groups of mice wereinjected subcutaneously with 1×10⁹ yeast cells in PBS. At various timepoints, the clearance rate of yeasts from the oral mucosa was determinedby swabbing the entire oral cavity. The swabs were resuspended in PBSand then serial dilutions of the cell suspension were plated onSabourand dextrose agar. The results (mean±SEM) were expressed as log10CFU per mouse. As shown in FIG. 7, mice immunised orally with eitherblastospores or hyphae rapidly cleared the yeasts compared withnon-immunised control mice. The clearance rate at various time pointsfrom mice immunised with blastospores was more rapid than from miceimmunised with hyphae forms. By comparison, mice immunisedsubcutaneously have poor clearance rate although by day 15, the yeastswere eliminated but not in unimmunised control, suggesting a mechanismof resistance involving the production of antibody as opposed to cellmediated immunity by oral immunisation where a rapid elimination ofyeasts occurred.

Example 8 IFN-γ and NO production following infection by Candidaalbicans and after Oral Immunisation

[0095] To identify the immune parameters of protection, the levels ofIFN-γ were determined at various times following infection with C.albicans. In FIG. 8 the effect of oral immunisation on saliva levels ofIFN-γ is shown. High levels are present before infection (day=0), 2 daysbefore similar levels are attained in control mice following oralinfection. This demonstrates that oral immunisation induces high levelsof IFN-γ in saliva, protecting against infection.

[0096] Since nitric oxide (NO) production is associated with hostdefence in parasitic infection, quantitation of NO was performedfollowing infection in two mouse strains sharing the same H2d MIChaplotype. In this experiment, mice were infected with C. albicans andthen followed by ip injection with an inhibitor of NO synthase, byinjecting NG-monomethyl-L-arginine monoacetate (MNLA) daily for 3 daysafter which time the clearance rate of yeasts was determined. As shownin FIG. 9, mice treated with MNLA had delayed clearance of yeasts atvarious time points in the two mouse strains compared with untreatedmice, indicating that reduction in NO production is associated withresistance.

EXAMPLE 9 Effect of Candida Vaccine Against Live Challenge with Candidaalbicans in the Oral Mucosa When Administered by the Oral Route.

[0097] DBA/2 male mice ( 6-8 weeks old) were immunised orally with 1×10⁸heat killed blastospores (Candivax) five times every 2 days for 10 daysor by subcutaneous injection of 1×10⁶ blastospores four times every 2days and then boosted on day 14 prior to live challenge with C albicans.Mice immunised by the oral route were better protected than miceimmunised by subcutaneous injection ( FIG. 10). The data are consistentwith the concept of a common mucosal immune system in that immunisationby the mucosal route is more effective against infection at mucosal sitecompared to immunisation by systemic route. However, mice immunisedorally with the sonicates of live organisms were less protected ( FIG.11).

EXAMPLE 10 Effect of Candida Vaccine on T Cell Proliferative Response inthe Regional Lymph Node

[0098] DBA/2 mice were orally immunised with Candivax which contained1×10⁸ blastospores PBS on 10 consecutive occasions every 2 days for 20days. After 8 weeks, one group of mice was given an oral boost one weekbefore challenge with C. albicans in the oral cavity. Control mice werefed PBS. After challenge, groups of mice were sacrificed at days, 2, 6and 8. The proliferative response of T cells was determined in cervicallymph node cells in culture stimulated with Candida antigens. After 3days in culture, the proliferative response was measured by tritriatedthymidine uptake. As shown in FIG. 12, T cell proliferation was higherin mice immunised with Candivax or Candivax plus booster compared withcontrol mice. Furthermore, mice given Candivax alone gave a betterresponse than mice given the vaccine plus an oral boost. In both cases,however, mice were protected from live challenege.

EXAMPLE 11 Therapeutic Effect of Candivax on Oral and GastrointestinalCandidiasis

[0099] Two groups of DBA/2 male mice ( 6-8 weeks old ) were infectedwith 1×10⁸ C albicans in the oral cavity. On day 2, one group wasimmunised daily on five consecutive days with 1×10⁸ autoclave heatkilled C. albicans blastospores in 200 microlitres of PBS, and thecontrol group was dosed with 200 microliters of PBS only.

[0100] Mice (groups of four) were sacrificed at days 4,6,8,12,15following oral infection and patterns of oral and gut C. albicansinfection were determined. Briefly, groups of mice were sacrificed atvarious time points as above. The oral cavity was completely swabbed.The yeast cells were resuspended by mixing on a vortex mixer beforeculture of serial 10-fold dilutions on Sabouraud dextrose agar plates.

[0101] In addition, the complete intestinal contents of each mouse wasremoved, suspended in 10 ml PBS, and after centrifugation at 400 g toremove luminal content, 10 ul of serial 10-fold dilutions were culturedon Sabouraud dextrose agar plates supplemented with chloramphenicol.After culture for 24 h at 37° C. the number of colonies were counted andthe number of C. albicans in the oral cavity and intestine determinedand expressed as CFU/mL.

[0102] The number of C. albicans recovered from the oral cavity is shownin FIG. 13, and the number of C. albicans in the intestine is shown inFIG. 14.

[0103] The results show a decrease in infection level in the oral cavityof the C. albicans-immunized group (compared to the control group) ondays 6 and 8, and a decrease in infection level in the intestine of theC. albicans group (compared to the control group) on days 8, 12 and 15.

[0104] This data shows that the C. albicans vaccine of the presentinvention has a therapeutic effect against an established C. albicansinfection. The data also shows that the vaccine composition of thepresent invention has a therapeutic effect against infection with Calbicans in the oral cavity but also in the gastrointestinal tract.

EXAMPLE 12 Effect of Administration of L. acidophilus on the Efficacy ofCandida Vaccine Against Oral Candidiasis

[0105] DBA/2 mice (6-8 weeks old) were administered 1×10⁸ L. acidophilus(VRI011) with 1×10⁷ heat-killed Candida vaccine or PBS by the oral routeevery other day for 20 days. One day after the final dose the mice werechallengedwith C. albicans in the oral cavity. At days 0, 2, 6 and 10,groups of mice were sacrificed and the level of colonisation in the oralcavity was determined. L. acidophilis (VRI011) can be sourced fromUniversity of New South Wales, School of Microbiology and ImmunologyCulture Collection, Sydney, Australia. However, a number of othercommonly used sources for Lactobacilli and other organisms will be knowto those skilled in the art

[0106]FIG. 15 shows that mice administered with L. acidophilus andCandida vaccine were significantly better in protecting against oralinfection with C. albicans (p<0.05) than Candida vaccine alone comparedwith control.

EXAMPLE 13 Compositions of Candivax

[0107] Composition A.

[0108] This is a monovalent oral killed candida vaccine conisisting of1×10⁸ killed C. albicans blastosphores. C. albicans (isolate 3630,National Reference Laboratory, Royal North Shore Hospital, Sydney,Australia) were cultured in Sabouraud dextroce broth (Oxid, UK) for 48hrs at 25° C. in a shaking water bath. The organisms were thentransferred into fresh medium and cultured at 25° C. a further 18 hrs.The blastosphores were collected by centrifugation 600 g for 10 mins at4° C., washed three times with PBS, resuspended in PBS and theninactivated by autoclaving at 121° C. for 30 mins. After autoclaving,the blastospores were washed three times in sterile PBS bycentrifugation, resuspended at 1×10⁹ cells per ml in Kreb's Ringerphosphate dextrose buffer ( KRPB) and then stored at 4° C. until use.The vaccine is stable for 6 months.

[0109] Composition B.

[0110] This is a combined oral killed Candida vaccine consisting of1×10⁷ heat killed C albicans blastospores and 1×10⁷ Lactobacillisacidophilus (VRI 011). Oral immunisation with the combined vaccine wasmore effective than either blastospores or L acidophilus alone (FIG.15).

[0111] The results of the above studies demonstrate that host resistanceto C. albicans infection in the oral mucosa in a murine model is linkedto a particular pattern of cytokine response and an accumulation of γ/δT cells in the regional lymph nodes. The differences in the colonisationpatterns of C. albicans in ‘infection-resistant’ BALB/c and‘infection-prone’ DBA/2 mice following infection correlated with both Tcell proliferation and the secretion pattern of the cytokines IL-4,IL-12 and IFN-γ. Colonisation patterns for both blastospore and hyphaeforms of C. albicans were cyclical with high levels of colonisation inDBA/2. The more ‘infection resistant’ BALB/c strain showed a single peakwith lower levels of colonisation and more rapid clearance of C.albicans from the oral cavity. There was a selective expansion of γ/δTcells in the regional lymph node, which correlated in time with theclearance of infection in both mouse strains. A sustained antigenspecific T cell proliferation was only produced in the infectionresistant BALB/c mouse strain. High levels of serum IgG and salivary IgAantibodies followed resolution of infection in BALB/c mice but to a lessor extent in DBA/2 mice. In DBA/2 mice, a cyclic colonisation with highnumbers of fungi, and delayed clearance of infection correlating withhigh early levels of IFN-γ and IL-12 following infection, but with adelayed and blunted ILK response. In contrast, the infection resistantBALB/c strain showed a single peak with low levels of colonisationfollowed by a rapid clearance of C. albicans , which also correlatedwith an early production of IL-4 and IFN-γ. Neutralization of IL-4 inthese mice by multiple injection of anti-IL-4 monoclonal antibody(11B11) resulted in an increase in carriage rate and a delayed clearanceof C. albicans from the oral cavity. Collectively, the results suggestthat the induction of a balanced Th1 and Th2 helper cell responsecharacterised by IFN-γ and IL-4 production, and the proliferation ofγ/δT cells, are factors associated with host resistance to C. albicansinfection in oral candidiasis.

[0112] The mechanisms of host protection against C. albicans infectionhave been extensively studied in murine models of candidiasis in termsof the impact of T cell cytokines operating through various effectormechanisms of immunity (10). In invasive candidiasis, neutrophils andmacrophages are involved in host defence (2). A link between resistanceand susceptibility, and T cell cytokine profiles, has been demonstratedin these models in terms of mortality or survival (reviewed in 2). Forinstance, IFN-γ is rapidly produced following infection in bothresistant and susceptible mice (28,44), neutralising IFN-γ increasedsusceptibility of resistant mice to infection (40) as was overproductionof IFN-γ mediated by IL-12 (28). In a study of IFN-γ deficient mice,IFN-γ induced activation of macrophages was essential for survival (24).Yet, other studies have shown that IFN-γ is not essential in hostdefence against systemic candidiasis (37). It is important todistinguish in such studies between mechanisms that limit mucosalcolonisation from those that prevent systemic invasion, and from thatessential for survival. Studies involving manipulation of singlecomponents of the host response while of value must be interpreted withcaution. The present study examined mechanisms of host resistance andsusceptibility in a natural model of self limited oral candidiasis.Different patterns of colonisation and IFN-γ and IL-4 production werecompared in ‘infection-resistant’ BALB/c mice and in an‘infection-prone’ DBA/2 strain. While IFN-γ transcripts was detectedearly (at 6 hrs) in both BALB/c and DBA/2 mice following an initialinfection with C. albicans , the production of IFN-γ did not on its ownprevent more protracted colonisation in DBA/2 mice. Whether deficiencyof the fifth component of complement in DBA/2 contributes to theprotracted colonisation in oral candidiasisis unclear. Several studieswith congenic mice including those bred from different geneticbackground of DBA/2 strain have reported that C5 deficiency is not anessential factor contributing to the pathogenesis of invasivecandidiasis since a reduction in inflammatory lesion at foci ofinfection was noted in C5-deficient DBA/2 mice (1, 2).

[0113] The present studies show that high levels of IL-12, IFN-γ and adelayed message expression and lower levels of IL-4 correlated withhigher levels of colonisation and a delayed clearance of C. albicans inDBA/2 mice. This is consistent with the observation which showed that C.albicans infection of the gastric mucosa in susceptible DBA/2 micecorrelates with decreased expression of IL-4 in Peyer's Patches (10). Bycontrast, the lower levels of IL-12, IFN-γ and early and higherproduction of IL-4 correlated with low colonisation and rapid clearanceof C. albicans in BALB/c mice suggest that the degree, the kinetics, andthe mix of cytokines may be critical factors in determining protectionafter challenge. Both Th1 and Th2 cytokines, albeit in different amountswith different kinetics of production, were present in DBA/2 and BALB/cmice recovering from oral candidiasis, as was seen in gastriccandidiasis (10). Thus resistance to primary infection with C albicansin the oral mucosa is associated with Th1 and Th2 responses.Furthermore, IL-4 appeared to play an important role in this process assuggested by the increased carriage rate and delayed clearance of Calbicans from the oral mucosa of BALB/c mice treated with anti-IL-4antibody.

[0114] The mechanism of IL-4 enhanced resistance to C albicans infectionin oral mucosa is unclear. In primary systemic candidiasis, IL-4 maylimit C albicans infection through promoting effector mediators ofimmunity including the differentiation of effector Th1 cells (31). Inparticular, IL-4 promotes the development of a protective Th1 responsein systemic and gastric candidiasis (10). Other studies have shown thatmice deficient in IL-4 were more susceptible to acute systemic infectionthan normal controls (32), but there was no difference in susceptibilityto orogastric candidiasis after challenge (49). These paradoxicalfindings may be explained by different experimental models which usedifferent mouse strains and different routes of challenge and doses ofC. albicans to induce systemic or mucosal candidiasis. For example,intragastric challenge with C. albicans induced a more severe gastriccandidiasis in BALB/c mice than in DBA/2 mice whereas the reverse wastrue for systemic candidiasis (10). In the current model, acute oralcandidiasis was induced by a topical application of C albicans asopposed to an intragastric challenge with a bolus of C. albicans toinduce orogastric candidiasis (32). Furthermore, topical application ofC albicans to the oral mucosa restricts the supply of antigen to gutassociated lymphoid tissue (GALT) compartment which may modify thecourse of infection via activation of the common mucosal immune system(14). Indeed, we have shown that oral immunisation with killed C.albicans resulted in lower colonisation and rapid clearance of yeastfrom the oral mucosa in DBA/2 mice.

[0115] In the present study both BALB/c and DBA/2 mice cleared infectionbefore the onset of antibody production, indicating that production ofserum IgG and secretory IgA antibodies did not play a significant rolein mucosal clearance. This is consistent with a study of murine gastriccandidiasis which showed both Th1 and Th2 cytokine production at thetime of clearance (10). Moreover, enhanced production of secretory IgAantibody, did not accelerate resolution of infection (10).

[0116] Despite an increase in cell counts in the CLN after infection,the relative proportions of CD4+,CD8+, α/β T cells and B cells remainedconstant suggesting cell recruitment rather than antigen-inducedproliferation of cells. However, there was a selective expansion of γ/δT cells, which correlated with the elimination of C. albicans . Whilethe numbers were low, the increase was significant considering thepaucity of γ/δT cells in peripheral lymphoid tissues (20). Increasednumbers of γ/δT cells have been reported after bacterial, viral andparasitic infections, suggesting a role for γ/δT cells in the first lineof host defence. It has previously been reported that increased numbersof γ/δT cells in the oral mucosa correlated with the pattern ofcolonisation in BALB/c and DBA/2 mice infected with C. albicans (12). Itis not clear, however, whether γ/δT cells are a source of IL-4. Althoughit has been reported that γ/δT cell clones and cell lines are capable ofsecreting this cytokine (4), we could not demonstrate significantamounts of IL-4 in γ/δ T cells in these mice (data not shown). It hasbeen recently reported that γ/δT cells can enhance nitric oxide (NO)production by macrophages in mice injected intraperitoneally with C.albicans (23), further linking γ/δT cells with mechanisms of resistance.Furthermore, NO can enhance IL-4 expression in T cells (15), whichfurther influences the balance of cytokine secretion. Thus mucosalcontainment of C albicans may depend on the interaction betweenmacrophages and T cells through the release of NO and IL-4 which hasbeen reported to enhance the killing of yeast cells by macrophages (19)bearing IL-4 surface receptors. γ/δT cells secrete IFN-γ and IL-4 whichboth activate macrophages and act directly on C albicans (38).Preliminary studies in the current model examining saliva levels of NOsupport this hypothesis.

[0117] In summary, analysis of regional lymph node cell populationsprovides data consistent with current ideas about cytokine function inexperimental models of infection. Without wishing to be bound by anyparticular mechanism of action, the findings presented in this study ofa model of oral candidiasis indicate that the production of IL-4 andIFN-γ, may be important to the resolution of mucosal infection in theintact animal. The early appearance of IL-4 production suggests theimportance of this cytokine in enhancing immunity against C. albicansinfection in the oral mucosa. The concurrent presence of high levels ofIL-12 and IFN-γ support the concept of a balanced Th1 and Th2 responseas being an efficient host defence mechanism in clearing oral mucosalinfection.

[0118] Although the present invention was described with reference tospecific examples and preferred embodiments, it will be understood thatvariations in keeping with the broad concepts and the spirit of theinvention herein described are also contemplated.

REFERENCES

[0119] 1. Ashman, R. B. 1997. Genetic determination of susceptibilityand resistance in the pathogenesis of Candida albicans infection. FEMS.Immunol. med. microbiol. 19: 183-189.

[0120] 2. Ashman, R. B.1998. Candida albicans:pathogenesis, immunity andhost defence. Res.Immunol. 149: 281-288.

[0121] 10. Chakir, J., L. Cote, Coulombe.C., and N. Delasriers.1994.Differential pattern of infection and immune response duringexperimental oral candidiasis in BALB/c and DBA/2 (H-2d) mice. OralMicrobiol. Immunol. 9: 88-94.

[0122] 12. Challacombe, S. J., and D. Rahman. 1994. Oral immunizationagainst mucosal candidiasis in a mouse model., Academic press.,Prague.

[0123] 14. Cho, Y. S., and H. Y. Choi.1979. Opportunistic fungalinfection among cancer patients. A ten year autopsy study. Am.J. Cln.Pathol. 72: 617-.

[0124] 17. Feldman, G. M., and D. S. Finbloom.1990. Induction andregulation of IL 4 receptor expression on murine macrophage cell linesand bone marrow derived macrophages by IFN-g. J.Immunol. 145: 854-859.

[0125] 19. Hurtrel, B., P. H. Lagrange., and J. C. Michel. 1980.Systemic candidiasis in mice. Tow main role of poly morphonuclearleukocytes in resistance to infection. Ann.Immunol. 131: 105-111.

[0126] 20. Jones-Carson, J., A. Vazquez-torres, Van der Heyde., T.Warner, R. Wagner, and E. Balish. 1995. gd T cell-induced nitric oxideproduction enhances resistance to mucosal candidiasis. Nat. Med. 1:552-557.

[0127] 23. Kaufmann, S. 1996. gd and other unconventional T lymphcytes:What do they see and what do they do? Proc. Natl. Acad. Sci. USA. 93:2272-2279.

[0128] 24. Klein, J. 1996. Whence the intestinalintra epitheliallymphocyte? J. Exp. Med. 184:1203-1206.

[0129] 28. Mencacci, A., G. Del Sero, E. Cenci, s. D, C. F.,, A. Bacci,C. Montagnoll, M. Kopf, and L. Romani. 1998. Endogenous interleukin 4 isrequired for development of protective CD4+ T helper type 1 cellresponses to candida albicans. J. Exp. Med. 187: 307-317.

[0130] 31. Odds, F. c. 1988. Candida and candidosis, 104-110. UniversityPark Press,Baltimore, Md.

[0131] 32. Puccetti, P., A. Mencacci, F. Cenci, R. Spaccapolo, P. Mosci,K. H. Enssle, L. Romani, and F. Bistini. 1994. Cure of murinecandidiasis by recombinant soluble IL-4 receptor. J. Infect. Dis. 169:1325-1331.

[0132] 37. Romani, L., A. Mencacci, U. Grohmann, S. Mocci, P. Puccetti,and F. Bistoni. 1992. Neutralizing antibody to interleukin 4 inducessystemic protection and T helper type 1-associated immunity in murinecandidiasis. J. Exp. Med. 176:19-25.

[0133] 38. Romani, L., A. Mencacci, E. Cenci, R. Spaccapelo, P. Mosci,P. Puccetti, and F. Brstoni.1993. CD4+ subset expression in murinecandidiasis. Th responses correlate directly with genetically determinedsusceptibility or vaccine-induced resistance. J.Immunol. 150: 925-931.

[0134] 40. Romani, L., A. Mencacci, L. Tonnetti, R. Spaccapelo, E.Cenci, P. Puccetti, S. F. Wolf, and F. Bistoni. 1994. Interleukin-12 isboth required and prognostic in vivo for T helper type-1 differentiationin murine candidiasis. J. Immunol. 152: 5167-5175.

[0135] 42. Romani, L., F. Bistoni, A. Mencacci, E. Conci, R. Spaccapolo,and P. Puccetti. 1996. IL-12 in candida albicans infections. Res.Immunol. 146: 532 538.

Claims defining the invention are as follows: 1 Composition suitable fororal administration, the composition including inactivated Candidaalbicans, for prophylactic or therapeutic treatment of a conditioncaused by Candida albicans colonisation and/or infection at a mucosalsurface. 2 Composition suitable for oral administration, the compositionincluding blastococcoid form of Candida albicans, for prophylactic ortherapeutic treatment of a condition caused by Candida albicanscolonisation and/or infection. 3 A composition according to claim 1,wherein the condition is selected from an oral, nasopharyngeal orrespiratory tract colonisation and/or infection by Candida albicans. 4 Acomposition according to any one of claims 1 to 3, in the form of avaccine 5 A composition according to any one of claims 1 to 4, whichincludes whole Candida albicans. 6 A composition according to any one ofclaims 2 to 5, which includes inactivated Candida albicans. 7 Acomposition according to any one of claims 1 to 6, further including apharmaceutically acceptable solvent, excipient, adjuvant or carrier. 8 Acomposition according to claim 7, wherein the adjuvant is selected toinduce a Th1 response 9 A composition according to any one of claims 1to 8, further including a probiotic. 10 A composition according to claim9, wherein the probiotic is a probiotic bacterium 11 A compositionaccording to claim 10, wherein the probiotic bacterium is a lactic acidbacterium. 12 A composition according to claim 11, wherein the lacticacid bacterium is Lactobaccillus acidophilus. 13 Method of prophylacticor therapeutic treatment of a condition caused by Candida albicanscolonisation and/or infection at a mucosal surface, including theadministration to a subject requiring such treatment of a compositionaccording to any one of claims 1, or 3 to
 12. 14 Method of prophylacticor therapeutic treatment of a condition caused by Candida albicanscolonisation and/or infection, including the administration to a subjectrequiring such treatment of a composition according to any one of claims2 to
 12. 15 Method according to claim 13 or claim 14, wherein thecondition is mucositis. 16 Method according to claim 13 or claim 14,wherein the condition is selected from group consisting ofrecent/persistent stomatitis, recurrent vulvovaginal candidiasis,oesophagitis and lower urinary tract or bowel colonisation. 17 Methodaccording to any one of claims 13 to 16, further including theadministration of an adjuvant. 18 Method according to claim 17, whereinthe adjuvant is selected to induce a response 19 Method according to anyone of claims 13 to 16, further including a probiotic. 20 Methodaccording to claim 19, wherein the probiotic is a probiotic bacterium.21 Method according to claim 20, wherein the probiotic bacterium is alactic acid bacterium. 22 Method according to claim 21, wherein thelactic acid bacterium is Lactobaccillus acidophilus. 23 Method accordingto any one of claims 13 to 22, wherein the adjuvant and/or the probioticis admininstered orally. 24 Method according to any one of claims 13 to22, wherein the adjuvant and/or the probiotic is administeredparenterally. 25 Method according to any one of claims 13 to 22, whereinthe adjuvant and/or the probiotic is administered before, during orafter cessation of treatment with the compounds according to any one ofclaims 1 to
 6. 26 Method according to claim 25, wherein the adjuvantand/or the probiotic is administered before treatment with the compoundsaccording to any one of claims 1 to
 6. 27 Method for monitoring Candidaalbicans vaccine requirement or Candida albicans vaccine efficacyincluding the measurement of IFN-γ, NO, and/or IL-14. 28 Method ofidentifying Candida isolates and/or Candida antigens effective as avaccine or as a vaccine component, including the measurement of IFN-γ,NO, IL-12 or IL-4 in a mouse model. 29 Method according to claim 27 orclaim 28, wherein the measurements is performed on a saliva sample. 30Method according to claim 27 or claim 28, wherein the measurements isperformed on blood sample. 31 Method according to claim 27 or claim 28,wherein the measurements is performed on a tissue sample. 32 Methodaccording to claim 31, wherein the tissue sample is obtained fromlymphoid tissue. 33 Method according to claim 32, wherein the sample isobtained from a lymph node. 34 Method according to any one of claims 27to 33, wherein the measurement of IFN-γ, NO, IL-12 and/or IL-4 consistsin determining the proportion of cells which express IFN-γ, NO, IL-12and/or IL-4.